Osteogenetic differentiation: a novel role of Slug protein

The regeneration of bone tissue depends on the concerted actions of a plethora of signals that recruit mesenchymal stem cells for lineage-specific differentiation. The signals are conveyed in hormones, growth factors and transcription factors. These molecules are crucial for the osteoblast commitment, differentiation, functions and, consequently, ensure the proper bone modelling and remodelling. Among these factors, Wnt proteins have a critical role in bone development and homeostasis. Accumulated evidences have shown that lymphocyte enhancer binding factor 1/T cell factor (Lef1/Tcf) transcription factors, the nuclear effectors of the Wnt/β-catenin signaling pathway, influence osteoblast proliferation, function, and regeneration. Nevertheless, most downstream bone-specific target genes of this pathway are only partially known. Among these, Slug has been recently implicated in osteosarcoma progression as a Wnt-responsive molecule strongly correlated with a loss of tumor suppressors such as E-cadherin. Slug, also named Snail2, belongs to the Snail family of genes encoding zinc-finger transcription factors. It is expressed at different stages of development in different tissues, mediates epithelial–mesenchymal transition and directs cell motility during embriogenesis. Slug is also expressed in most normal adult human tissues, but little is known about its potential functions. In order to identify new potential osteoblast-specific proteins, in this study we analysed the expression, regulation and role of Slug in human normal primary osteoblasts (hOBs) and in their mesenchymal precursors (hMSCs), in relation to the expression of Wnt/β-catenin signalling mediators and genes which are required in the control of osteochondroprogenitors differentiation. The experiments were performed on hOBs and hMSCs, obtained from bone marrow iliac crest, bone marrow tibial plateau and Wharton’s jelly umbilical cord. Using several molecular analysis including siRNA strategy and Chromatin Immunoprecipitation (ChIP) assay, we demonstrated that: - Slug is expressed in hOBs as well as their mesenchymal precursors; - In hOBs, Slug is regulated by β-catenin and Lef1 that, together with Tcf-1, Tcf-4 and Runx2 are recruited to the Slug gene promoter in vivo; - In hOBs, Slug is positively correlated with osteoblastic markers, such as Runx2, osteopontin, osteocalcin, collagen type I, CXCL12, Wnt/β-catenin signalling and mineral deposition. At the same time, it negatively correlated with Sox9, a factor indispensable for chondrogenic development; - In hMSCs, Slug acts as a negative regulator of Sox9 and Sox5 expression and a positive regulator of Sox6 and STAT1 genes. Regarding Runx2, the role of Slug seems influenced by cell type; - Slug interacts in vivo with Runx2 and Sox9 promoters in hOBs and hMSCs. Our results support the hypothesis that Slug functions as a novel regulator of osteoblast activity, even if with a different role in mature committed osteoblasts and in their undifferentiated progenitors. Furthermore, these findings suggest Slug as a new potential therapeutic target for bone tissue repair and regeneration.